1. Field of the Invention
This invention relates to diagnostic assays, and more particularly to diagnostic assays which utilize peroxides and wherein a microperoxidase is utilized advantageously, in place of an enzyme, as a reagent.
2. Background Art
The use of enzymes as reagents is now widely recognized, especially in clinical determinations of the presence and/or concentration of a variety of analytes of interest and clinical significance in biological fluids, for example, blood serum, plasma, whole blood, urine, spinal and amniotic fluids. As used herein, an xe2x80x9cenzymexe2x80x9d is defined as a polypeptide having a molecular weight greater than about 10,000 daltons and exhibiting catalytic activity. In general, two basic approaches have been used in the art with respect to such enzymatic assays. In one, a precursor or substrate of clinical importance is converted by an enzyme into detectable compounds, or signals, such as color or fluorescence, which are detectable. In the second, the enzyme itself is coupled with an analyte and the enzyme activity is determined as a measure of the analyte present. Whichever approach is utilized, however, the enzyme reagent is generally required to possess good stability, be readily commercially available, and as well have what is termed in the art a xe2x80x9chigh turnover numberxe2x80x9d. In addition, it is desirable that the enzyme be active with respect to substrates which are readily commercially available, relatively stable and inexpensive and which produce easily detectable products or signals.
Typical of enzymes commonly used in assays such as the aforedescribed are redox enzymes, kinases and esterases. One example of the conventional use of enzymes in clinical chemistry assays is in the determination of uric acid, a procedure which has typically employed horseradish peroxidase (HRPO). In that analysis, uric acid is determined using HRPO in conjunction with uricase enzymes. Typical clinical test samples contain uric acid in concentrations less than about 12 milligrams per deciliter (mg/dl), and are contacted in such assays with microbial uricase which converts the uric acid to allantoin and hydrogen peroxide. The hydrogen peroxide thus formed is used to oxidize chromogenic substrates catalyzed by the HRPO, which substrates develop color as a measure of the presence and/or concentration of uric acid in the sample. The color development can then be measured visually, spectrophotometrically or by other instrumental means, and its intensity correlated with the amount of uric acid in the sample.
However, the use of HRPO or other enzymes in such conventional assays is not without problems. For example, the pH values for optimum activity of enzymes such as HRPO and uricase are quite different, and thus neither can be utilized efficiently in such assays. Because uricase is an expensive enzyme, the reaction conditions of such assays ordinarily have been adjusted to facilitate the most efficient use of uricase; such adjustment, in turn, requires concommitant use of a high concentration of HRPO to achieve a rapid kinetic response. In addition, HRPO is known to lose activity in storage, and hence has a limited shelf life.
Another conventional use of HRPO is as an enzyme label in immunoassays. For example, a number of known enzyme immunoassays involve contacting samples suspected of containing antigens of interest with a solid phase support containing or coated with an immobilized, adsorbed or covalently coupled specific antibody to the antigen. After incubation and washing of the solid phase, a conjugate, which comprises a specific antibody to the antigen which has been labeled with HRPO, is added and incubated with the solid phase. The solid phase is again washed, and the activity of the HRPO which becomes bound to the solid phase with the conjugate is then determined, usually by spectrophotometric means, by addition of an indicator substance comprising a chromogenic or other substrate. Upon contact with the HRPO, the indicator, if a chromogen, produces a color detectable by the spectrophotometer. The spectrophotometric reading can then be correlated with similar readings taken from known concentrations of the antigen, thereby to determine the concentration of antigen in the sample.
Horseradish peroxidase can also be disadvantageous in enzyme immunoassays of the type aforedescribed. For example, coupling conditions, well-known in the art, which ordinarily are used to produce the antibody-HRPO conjugate, have a tendency to inactivate both the antibody and the HRPO. Moreover, such coupling conditions usually tend to produce a heterogeneous product requiring extensive purification and careful optimization for each particular assay. Also, the conjugates produced have a marked tendency to become unstable during storage, limiting their shelf-life.
In accordance with the present invention, a method has been discovered for carrying out diagnostic assays which overcomes the aforedescribed disadvantages of conventionally-used enzymes. The method can be employed to particular advantage in determining various analytes in biological fluids, such as blood serum, whole blood, plasma, urine, spinal and amniotic fluids, and the like. The method utilizes a microperoxidase as a non-enzymatic catalyst, to provide such assays with advantageous stability and the capability of being used efficiently with enzymes to provide rapid kinetic responses to analytes.
In a preferred embodiment, the method which is improved by the invention comprises contacting a sample containing an analyte, or a derivative thereof, which is of interest with an oxidoreductase enzyme to produce a peroxide reaction product; the reaction product, in the presence of a peroxidease and a substance capable of producing a detectable response thereto, can then be determined as a measure of the presence and/or amount of the analyte in the sample. The improvement of the invention comprises using, as the peroxidase, a microperoxidase.
The concepts of the present invention reside in an improved assay method for determining the presence of an analyte in a test sample, such as blood serum or plasma, in which an oxidoreductase and a peroxidase, upon contact with the analyte or a derivative thereof in the sample, produces a reaction product which can be determined as a measure of the presence and/or amount of the analyte in the sample. specifically, it has been discovered that in the practice of the method of the invention, a microperoxidase functions similarly to conventionally-used HRPO or other enzymes in typical examples of such assays and, therefore, microperoxidase can be directly substituted for such enzymes which would otherwise be used in such assays, but with the various advantages over the use of such enzymes which are detailed herein.
Microperoxidases are non-enzymatic, catalytic entities which, in accordance with the invention, have been found to provide peroxidative activity in assays such as the aforedescribed. Moreover, when a microperoxidase is used at or near the optimum pH of oxidaoreductase enzymes or reagents used in the assay in conjunction with the microperoxidase (e.g., uricase in uric acid assays) more efficient use of the relatively expensive uricase or other enzymes or reagents can be achieved. In addition, at moderate concentrations, the use of a microperoxidase has been found to provide rapid kinetic responses, and microperoxidases have also been found to be very stable under clinical assay conditions. Moreover, it has been determined that microperoxidases can be readily coupled to antigens, and antibodies, to provide advantageously stable conjugates for use in both heterogeneous and homogeneous immunoassay systems. Microperoxidases, in accordance with the invention, have also been found to provide advantageous performance in non-immunoassay systems wherein peroxidases have conventionally been used.
As is known to those skilled in the art, microperoxidases are fragments of cytochrome C produced by enzymatic cleavage of the protein. Such cleavage leaves small sections of the original cytochroine C amino acid chain with its heme group covalently attached through thioether bonds to the peptide. The structure of compounds characterized as microperoxidases can be generally represented as follows: 
Various microperoxidases are commercially available which are suitable for use in the present invention. For example, the microperoxidase designated MP-8, having a molecular weight of 1502, and microperoxidases MP-9 and MP-11, having molecular weights of 1630 and 1857, respectively, are all suitable and substantially equivalent for use in the invention and are commercially available, for example from Sigma Chemical Company.
Microperoxidases, in accordance with the concepts of the present invention, are particularly well suited for use in place of HRPO in uric acid assays. In a preferred practice of the invention, as set forth in more detail infra, a clinical test sample containing uric acid, usually at concentrations below 12 milligrams per deciliter (mg/dl), is contacted with a mixture of uricase enzyme and microperoxidase in the presence of one or more oxidizable or chromogenic substrates which develop color on oxidation. Such substrates which are useful in the present invention for assay of uric acid as well as other analytes are known in the art, and include, for example, O-phenylenediamine (OPD), 2,2xe2x80x2-azino-di-(3-ethylbenzothiazylene sulfuric acid) (ABTS), 3,3xe2x80x2,5,5xe2x80x2-tetramethylbenzidine (TMB), 4-aminoantipyrene (4AAP) and 2-hydroxy-3,5-dichlorobenzene sulfonic acid (DHBS). Such compounds are capable of reaction with peroxides in the presence of microperoxidase to produce a detectable response, such as the development of color on oxidation, as a measure of the peroxide present, and hence as a measure of the uric acid present in the sample. In the practice of the invention, such color development can be conveniently determined in accordance with conventional techniques, and is preferably determined spectrophotometrically using conventional analytical instrumentation, to provide a quantitative measurement of the analyte in the test sample. Alternatively, the color response can, for example, be determined visually, as when it is only desired to use the assay as a semiquantitative screen test for the presence of the analyte.
In addition, luminol or its isomers, for example, can be employed as the substrate for chemiluminescent detection, or various fluorescent compounds such as flourescein or its derivatives can be used as substrates, if desired, to produce a measurable fluorescent response which can be correlated with the presence and/or amount of analyte in a sample under analysis.
In addition to use in the determination of uric acid, it is to be appreciated that a microperoxidase, in accordance with this invention, can also be used as a catalytic unit for the conversion of hydrogen peroxide formed by enzymatic reactions with a number of other compounds, notably including glucose, cholesterol and triglycerides. Thus, those compounds will produce hydrogen peroxide under the action of various known enzymes and reagents, and the hydrogen peroxide so formed can be measured using microperoxidase in a calorimetric or other assay similar to that previously described for uric acid.
In addition, a microperoxidase can, in accordance with the concepts of this invention, be coupled with antibodies to form a conjugate containing the microperoxidase. Exemplary of this technique is a process wherein a sample containing an antigen is contacted with a solid phase such as a polystyrene bead coated with an antibody to the antigen, whereby the antigen becomes immobilized on the bead. After incubation and washing of the bead, a specific antibody to the antigen which has been labeled with microperoxidase is contacted with the bead, incubated, and later washed. The activity of the microperoxidase can then be determined spectrophotometrically or by other means as a measure of the concentration of the antigen in the sample, e.g., through the use of an oxidizable chromogenic substrate, described supra.
A wide variety of antibodies can be coupled with a microperoxidase using homo and hetero bifunctional coupling reagents. Such antibodies include, for example, the monoclonal antibody to Carcino Embryonic Antigen (CEA). In addition, a microperoxidase can also be employed in the instant invention in the labeling of antigens, for example in homogenous immunoassays, and microperoxidases can be employed, using conventional techniques, to label drugs such as theophylline as well as variety of other antibodies and antigens, in accordance with known techniques.
It has also been found, in accordance with the concepts of the present invention, that the sensitivity of an assay utilizing a microperoxidase can be increased by covalently coupling the microperoxidase to polymeric molecules, including for example, dextrans or polyamino acids. Alternatively, the microperoxidase can be coupled with itself. Such conjugates and multiple microperoxidase molecules can then be coupled and used in an assay as previously described.